Detergents comprising cellulases

ABSTRACT

A laundry detergent composition comprises a cellulase having a ratio of tensile strength loss to antipilling properties of less than 1. A method of laundering cotton-containing fabrics with the composition is also disclosed.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the use of novel cellulases withimproved properties in detergents and aqueous laundry solutions. Theinvention further relates to detergents and detergent additivescomprising the novel cellulase.

2. Discussion of the Related Art

Cellulases, also called cellulolytic enzymes, are enzymes which arecapable of the hydrolysis of the β-D-glucosidic linkages in celluloses.Cellulolytic enzymes have been divided traditionally into three classes:endoglucanases, exoglucanases or cellobiohydrolases and β-glucosidases(Knowles, J. et al. (1987), TIBTECH 5, 255-261). Cellulolytic enzymescan be produced by a large number of bacteria, yeasts and fungi.Microorganisms that produce cellulases are described in for exampleGB-A-2094826.

Several applications have been developed for the use of cellulolyticenzymes:

degrading (wood) cellulose pulp into sugars for (bio)ethanol production;

several textile treatments like ‘stone washing’ and ‘biopolishing’;

application in detergent compositions.

The use of cellulases in detergent compositions started with cellulasescapable of reducing the harshness, i.e. softening of cotton containingfabrics, as described in for example GB-B-1358599.

It is further known that detergent compositions comprising cellulasesare effective in removing dirt, i.e. cleaning. The efficiency ofcellulolytic enzymes, cellulases, in terms of cleaning textile has beenrecognized for some time. GB-A-2075028, GB-A-2095275 and GB-A-2094826disclose detergent compositions with cellulase for improved cleaningperformance.

It is also known in the art that cellulases can act as a colourclarifying agent in laundry detergents. After repeated washing of soiledfabrics, cotton containing fabrics appear to be greyish, most probablydue to disrupted fibres caused by mechanical action. The fibres are tornup resulting in disordered fibres which are broken. The use ofcellulases as colour clarification agents for coloured fabrics has beendescribed in EP-A-0220016. Actually cellulase mixtures from the fungalstrain Humicola insolens (DSM 1800) are commonly used in detergents toresult in antipilling and colour revival properties. The cellulolyticenzyme system produced by the wild type microorganism is available underthe trade name of Celluzyme® by Novo-Nordisk. In addition a cloned(single) cellulase from the same origin under the trade name Carezyme®is also used in detergents.

The main disadvantage of the cellulases known in the art showing colourclarification is that these enzymes aggressively degrade the cellulosecontaining fabrics which results in damage by undesirable loss oftensile strength of the fabrics.

On the other hand cellulases known in the art showing good cleaningproperties show hardly any colour clarification effects. The firstcommercial detergent with cellulases in the world contained a bacterialcellulase. This enzyme represents an above mentioned alkalineendoglucanase from a Bacillus species that does not attack cellulosefibres. The enzyme is described to give a cleaning effect duringwashing. No effects with respect to anti-pilling or colour revival havebeen described for this enzyme.

From the above it will become clear that it is still desirable toprovide for improved cellulases in detergent applications. Usingmixtures of cellulases, as suggested in international patent applicationWO-A-95/02675, is supposed to provide the above mentioned performance inlaundry washing, but to our knowledge, it has not previously beenpossible to use single enzymes providing all these characteristics whenapplied in laundry washing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative activities of the cellulase BCE 103. InExample 1, this figure is referred to as the pH/temperature profiles.All activities for both 40° C. and 60° C. are related to the highestactivity fixed at 100%.

FIG. 2 shows the relative activities of the cellulase BCE 113.

DESCRIPTION OF THE INVENTION

Surprisingly it has been found that the use of certain single cellulaseswhich are capable of cleaning, antiredeposition, colour clarificationand antipilling performance in laundry washing does not at all result inunacceptable damage to the textiles washed.

Accordingly, the present invention relates to the use of a singlecellulase with a ratio of tensile strength loss (TSL, as herein defined)to antipilling properties (AP, as herein defined) below 1 in aqueouslaundry solutions.

To measure tensile strength loss is a way to measure damage caused bymechanical stress of enzymatical action on fibers. It is to beunderstood that for the purpose of the present invention cotton fiberhas to be used. The method measures the tensile strength of singlefibers under wet conditions. It is described in German Standard DIN 53,857, part 1 as well as in the International Standard ISO 2267.

As the effect normally shows up in a significant amount only after about20 to 25 wash cycles, there is always some tensile strength loss due tothe mechanical forces acting on the cotton fiber during the washingprocess. Therefore the tensile strength loss of a control fabric washedwithout cellulases using the same formulation of detergent and the sametype of washing machine and washing programme has to be subtracted. Tocalibrate the values, a preparation of the (single) endoglucanase V fromHumicola insolens (EG V) in equal amounts of enzymatic protein in thedetergent is used as a standard and the value of the tensile strengthloss for this sample minus the control value of detergent withoutcellulase is taken as a TSL of 100%. This cellulase EG V has beendescribed for example in the international patent application WO91/17243. The amount of protein can be measured for example by using theBCA Pierce method as described by R. E. Brown et al. in Anal. Biochem.1989, vol. 180, p. 136-139.

A preparation of an above mentioned Bacillus cellulase available fromKao Corp. under the trade mark KAC® 500 or KAC® 700 may be used ascomparison, resulting in general in a very low tensile strength loss ascompared to the control washing experiment with no cellulase present.

The attack of cellulases on protruding microfibrils, pills and cottonfluff on the surface of a cotton fabric results in an optically visibleremoval of that pills. To test the effect, washings are to be performedusing a detergent with and without cellulase, as described for thedetremination of TSL. The antipilling effect, too, can best be seenafter an increasing number of wash cycles. Therefore a number of 15 to40 wash cycles are generally used to demonstrate this effect ofcellulases.

There are three different methods that can be used for quantification ofthis effect:

1. visual evaluation by a test group (panel)

2. measurement of light reflection (L-value of the CIELAB-system)

3. determination of the cotton fluffs by means of optical measurement

The determination using the L-value of the CIELAB-system [CommissionInternationale de l'Éclairage] was described by U. Hotz in Tenside Surf.Det. 1993, vol. 30, page 388. The optical measurement system, which isused in the preferred method of determining the antipilling properties,usually consists of a light source, a microscope tube and a CCD colourcamera recording the light reflected from the surface of a fabric.Depending on the amount of pills and fluff on the surface of the fabricthe amount of reflected light as measured by digital image analysischanges. Such a system can be used to measure quantitatively the amountof pill and fluff on fabrics, normally after 15 to 40 wash cyclesdepending on the type and activity of the cellulase added to thedetergent. An optical system which can be used to measure the degree ofpilling has been described by T. Müller-Kirschbaum and H. Grundmann inSÖFW, vol. 118 (1992), p. 483-499.

Whatever method is used to determine the antipilling effect of thecellulase to be tested, the standard cellulase EG V has to be testedunder the same conditions and its effect has to be determined by thesame method, taking into account the value resulting from the use of thedetergent without cellulase. The value obtained for EG V is taken asAP=100%.

As can be seen from this definition, the known cellulase EG V fromHumicola insolens has a ratio of TSL to AP of 1. As the above mentionedBacillus cellulase available from Kao Corp. under the trade mark KAC®500 or KAC® 700 has a low AP and a very low TSL, it can be seen thatalso the ratio for this cellulase is approximately 1. Cellulases whichmay be used according to the invention, especially in detergents, have aratio of TSL to AP as much as possible below 1, preferably below 0.8 andmore particularly in the range of 0.001 to 0.5. A ratio of TSL to AP offor example 0.5 means that only 50% of tensile strength loss is seen atan enzyme concentration yielding the same antipilling effect as thestandard cellulase.

The aqueous laundry solution preferably comprises cellulase according tothe definition given above in concentrations of 0.01 mg/l to 0.2 mg/l,more particularly 0.015 mg/l to 0.1 mg/l. These concentrations refer tothe weight of cellulolytic protein. In addition all ingredients normallyfound in laundry solutions can be present.

Another aspect of the present invention is the use of a single cellulasewith a ratio of TSL to AP below 1 to provide an anti-greying effect tofabrics, especially coloured fabrics.

In another aspect of the invention a single cellulase with a ratio ofTSL to AP below 1 is used to provide a softening effect to fabrics.

The present invention also relates to the use of a single cellulase witha ratio of TSL to AP below 1 to provide colour clarification or toinhibit colour deterioration of fabrics, especially coloured fabrics.

The present invention further relates to the use of single cellulasewith a ratio of TSL to AP below 1 to inhibit the wrinkling of fabricsand to ease the ironing of fabrics.

We found that the use of a single cellulase according to the definitionof the invention, unlike previously known mixtures of cellulases whichprovide colour clarification, does not degrade cotton to an undesirablelevel causing tensile strength loss.

It is further found that in using a cellulase of the definitionaccording to the invention, unlike previously known cellulases whichprovide colour clarification, the enzyme does not accumulate on thefabric after repeated laundry washing.

In another aspect, the invention is directed to detergent compositions,detergent additives and fabric softener compositions comprising a singlecellulase according to the definition given above.

As noted before, the present invention generally relates to the use ofnovel cellulases. However, prior to disclosing this invention in moredetail, the following terms will be defined.

_(“)Cellulase” is a generic name for enzymes acting on cellulose and itsderivatives, and hydrolysing them into glucose, cellobiose orcellooligosaccharides.

The term _(“)single” cellulase used herein is intended to mean acellulase which is produced by one gene.

_(“)Obtainable form” an organism in connection with a cellulase meansthat such cellulase has an amino acid sequence which corresponds to theamino acid sequence of a cellulase which may be obtained from thatorganism.

_(“)Derivative” is intended to indicate a protein which is derived fromthe native protein by addition of one or more amino acids to either orboth the C- and N-terminal end of the native protein, substitution ofone or more amino acids at one or a number of different sites in thenative amino acid sequence, deletion of one or more amino acids ateither or both ends of the native protein or at one or more sites in theamino acid sequence, or insertion of one or more amino acids at one ormore sites in the native amino acid sequence. The preparation of aderivative is usually achieved by modifying a DNA sequence which encodesfor the native protein, transformation of that DNA sequence into asuitable host and expression of the modified DNA sequence to form thederivative protein. The derivative of the invention includes peptidescomprising altered amino acid sequences in comparison with a precursorenzyme amino acid sequence (e.g., a wild type or native state enzymeaccording to the present invention) and which peptides retain acharacteristic enzyme nature of the precursor enzyme but which havealtered properties in some specific aspect. For example, an alteredcellulase may have an increased pH optimum or increased temperatureresistance but will retain its characteristic cellulase activity.Derivatives also includes chemical modifications of amino acid residueswithin the enzyme molecule.

_(“)Host cell” means a cell which has the capacity to act as a host andexpression vehicle for a recombinant DNA vector comprising DNA whichencodes for the native protein or a derivative.

The term _(“)cleaning” means the removal of dirt attached to laundry.

The term _(“)pilling” in this respect is the formation of pills and fuzzon the surface of cotton containing fabrics due to broken or disorderedfibres.

The term _(“)antipilling” is used to describe the prevention of theformation of pills and fuzz on the surface of cotton containing fabricsas well as the removal of pills and fuzz from cotton containing fabrics.Antipilling normally results in colour clarification when colouredcotton containing fabrics are treated.

The term _(“)color clarification” in this respect is the reestablishmentof the attractive fresh look of coloured fabrics containing orconsisting of cellulose based fibres, which have developed a greyishappearance by treatment, especially with laundry detergents, of thecoloured fabric.

The term _(“)redeposition” in this respect is deposition of dirt orcolour components that were removed from these textiles or fabricsduring a laundry washing or textile treatment.

The term _(“)antiredeposition” in this respect is the action ofcellulase to prevent or diminish the redeposition of dirt and colourcomponents on the fabric.

By a _(“)laundry solution” is meant an aqueous solution used forwashing, rinsing or conditioning, e.g. softening, fabrics.

In a preferred aspect, the present invention relates to the use of acellulase which is obtainable from microorganisms which are depositedaccording to the Budapest Treaty on the International Recognition of theDeposits of Microorganisms for the Purposes of Patent Procedures, at theCentral Bureau voor Schimmelcultures, Baam, The Netherlands on Dec. 23,1993 under deposition numbers CBS 669.93 and CBS 670.93 (described ininternational patent application WO-A-95/18219). This strains have beenclassified as new species of the genus Bacillus, which do not belong toany of the presently known rRNA-groups of Bacillus. As used herein, thedeposited species will be referred to as CBS 669.93 and CBS 670.93.

The microorganisms may be obtained for example from water and soilsamples collected in alkaline environments such as alkaline soils andsoda lakes.

The microorganisms have subsequently been screened using a carboxymethylcellulose (CMC)-agar diffusion assay. Strains which showed a clearingzone in this test were isolated as potential cellulase producingstrains. Genomic gene libraries of the alkali tolerant cellulaseproducing strains were constructed. Recombinant clones were screened byagar diffusion on CMC-agar. Recombinant clones that showed clearingzones around the colony were isolated. Single cellulases were producedby fermentation of the recombinant clones in 4*YEP-medium for 48 hoursat 30° C. The obtained single cellulases, optionally purified asdescribed in Example 1, were tested in the tests defined above tomeasure TSL and AP.

Surprisingly it was found that the cellulase obtainable from CBS 670.93or CBS 669.93 show a good performance in both tests and have a ratio ofTSL to AP below 1.

In a preferred embodiment of the invention, an approximately 50 kDcellulase (calculated on the basis of the amino acid sequence (SEQ IDNO:1) of the mature protein) derived from CBS 670.93 (referred to as_(“)BCE 103” herein) is used. It has been revealed by analyzing the geneencoding the amino acid sequence of the approximately 50 kD cellulasethat this cellulase is 89% identical in sequence and 92.5% similar insequence to the cellulase CelA of Bacillus sp. N-4 (Fukumori et al., J.Bacter., vol. 168, pp. 479-485) by using the TFastA program (SequenceAnalysis Software Package 6.0 of Genetic Computer Group, University ofWisconsin, Biotechnology Center, Madison, Wis.) as described by Pearsonand Lipman in Proc. Nat. Acad. Sci., vol. 85, pp. 2444-2448 (1988). Theamino acid sequence of BCE 103 is given in SEQ. ID. NO:1. The presentinvention further encompasses the use of cellulases with an amino acidsequence which have greater than 89%, preferably greater than 95%sequence identity and/or greater than 92.5%, preferably greater than 97%sequence similarity thereto, and detergents comprising such a cellulase.

In an equally preferred embodiment of the invention, an approximately 63kD cellulase (calculated on the basis of amino acid sequence of themature protein) derived from CBS 669.93 (referred to herein as _(“)BCE113”) is used. It has been revealed by analyzing the gene encoding theamino acid sequence of the approximately 63 kD cellulase that thiscellulase is 58% identical in sequence and 72% similar in sequence tothe cellulase CelB of Bacillus lautus (Jorgensen et al, Gene, vol. 93(1990), p. 55-60) by using the TFastA program (Sequence AnalysisSoftware Package 6.0 of Genetic Computer Group, University of Wisconsin,Biotechnology Center, Madison, Wis.) as described by Pearson and Lipmanin Proc. Nat. Acad. Sci., vol. 85 (1988), p. 2444-2448. The amino acidsequence of BCE 113 is given in SEQ ID NO:2. The present inventionfurther encompasses the use of cellulases with an amino acid sequencewhich have greater than 58%, preferably greater than 80% and moreparticularly greater than 90% sequence identity and/or greater than 72%,preferably greater than 80% and more particularly greater than 90%sequence similarity thereto, and detergents comprising such a cellulase.A cellulase which may be used in detergents according to the presentinvention in addition to having a ratio of TSL to AP below 1 usuallyperforms well in the Antiredeposition Test as described in Example 4.Whiteness maintenance of white fabric is measured by a reflectancemeasurement. The higher the reflectance value, the more effective thetested cellulase is in antiredeposition performance. They also performwell in the Softening Test as described in Example 4. Depilling is theremoval of fibrils and/or microfibers that are disordered and/or brokenwhich usually make a coloured cotton containing fabric look greyish. Themore disordered and/or broken fibrils are removed the better thecoloured cotton containing fabrics look. Depilling effectiveness can bejudged by panels or can be quantified by an image analysis system, asspecified above for the measurement of AP. Cellulases which fulfil therequirement of the ratio defined above usually exhibit the followingproperties: They show a delta REM of at least 4 units, preferably atleast 5 units, in the Anti Redeposition Test as defined in the Examples,and they show a depilling result which is at least comparable to that ofthe cellulase obtainable from CBS 670.93.

The cellulases which can be used according to the present invention maybe produced by a process which can be developed using geneticengineering. As a first step the gene encoding the cellulase of thepresent invention can be cloned using λ-phage (expression) vectors andE. coli host cells. Alternatively PCR cloning using consensus primersdesigned on conserved domains may be used. Expression of the geneencoding the cellulase of the present invention in E. coli is shown togive an active protein.

After a first cloning step in E. coli, a cellulase gene can betransferred to a more preferred industrial expression host such asBacillus or Streptomyces species, a filamentous fungus such asAspergillus, or a yeast. High level expression and secretion obtainablein these host organisms allow accumulation of the cellulase of theinvention in the fermentation medium from which they can subsequently berecovered.

Cellulases according to the invention are preferably used in amounts of8·10⁻⁵% by weight (0.8 ppm) to 8·10⁻³% by weight (80 ppm), moreparticularly 1·10⁻⁴% by weight (1 ppm) to 4·10⁻³% by weight (40 ppm),referring to the celluloytic protein, in detergents. Detergentcompositions comprising a cellulase defined according to the inventionmay additionally comprise surfactants which may be of the anionic,non-ionic, cationic, amphoteric or zwitterionic type as well as mixturesof these surfactant classes. Detergent compositions of the invention maycontain other detergent ingredients known in the art, as e.g. builders,bleaching agents, bleach activators, anti-corrosion agents, sequesteringagents, soil release polymers, perfumes, other enzymes, enzymestabilizers, etc.

Suitable binders are in particular those from the classes ofpolycarboxylic acids, more particularly polymeric acrylic acids,methacrylic acids, maleic acids, copolymers thereof and oxidizedcarbohydrates, as described in international patent applicationWO-A-93/16110, layer silicates, more particularly bentonites,alumosilicates, more particularly zeolites, crystalline or amorphousalkali metal silicates, more particularly sodium silicates, and alkalimetal carbonates, more particularly sodium carbonate. The polycarboxylicacids mentioned are normally used in the form of their alkali metalsalts, more particularly in the form of their sodium or potassium salts.The zeolites preferably incorporated are in particular those of the A, Por X type or mixtures thereof. Preferred alkali metal silicates arethose with molar ratios of SiO₂ to alkali metal oxide of 1.5 to 3.0.Builders such as these are preferably present in detergents according tothe invention in quantities of 20% by weight to 80% by weight.

Nonionic surfactants may be present in the detergents according to theinvention, preferably in quantities of not more than 10% by weight and,more preferably, in quantities of 2% by weight to 6% by weight, based onthe detergent as a whole. Suitable nonionic surfactants are alkylpolyglycosides containing 10 to 22 carbon atoms in the alkyl componentand alkoxylates, particularly ethoxylates and/or propoxylates, of linearor branched C₁₀₋₂₂ and preferably C₁₂₋₁₈ alcohols. The degree ofalkoxylation of the alcohols is between 1 and 20 and preferably between3 and 10. They may be prepared in known manner by reaction of thecorresponding alcohols with the corresponding alkylene oxides. The fattyalcohol derivatives are particularly suitable, although theirbranched-chain isomers, more particularly so-called oxoalcohols, may beused for the production of useable alkoxylates. Accordingly, theethoxylates of primary alcohols containing linear dodecyl, tetradecyl,hexadecyl or octadecyl radicals and mixtures thereof are particularlyuseful. In addition, corresponding ethoxylation and/or propoxylationproducts of alkyl amines, vicinal diols and carboxylic acid amides,which correspond to the alcohols mentioned in regard to the alkylcomponent, and of alkyl phenols containing 5 to 12 carbon atoms in thealkyl component may also be used. Suitable anionic surfactants are inparticular those of the sulfate or sulfonate type, although other types,such as soaps, long-chain N-acyl sarcosinates, salts of fatty acidcyanamides or salts of ether carboxylic acids, which may be obtainedfrom long-chain alkyl or alkyl phenyl polyglycol ethers and chloroaceticacid, may also be used. The anionic surfactants are preferably used inthe form of the sodium salts. Surfactants are preferably present inquantities of 2% by weight to 30% by weight and more preferably inquantities of 5% by weight to 20% by weight.

Particularly suitable surfactants of the sulfate type are the sulfuricacid monoesters of long-chain primary alcohols of natural and syntheticorigin containing 10 to 20 carbon atoms, i.e. the sulfuric acidmonoesters of fatty alcohols such as, for example, coconut oil fattyalcohols, tallow fatty alcohols, oleyl alcohol, or the C₁₀₋₂₀oxoalcohols and those of secondary alcohols of the same chain length.The sulfuric acid monoesters of aliphatic primary alcohols, secondaryalcohols and alkyl phenols ethoxylated with 1 to 6 mol ethylene oxideare particularly suitable. Sulfated fatty acid alkanolamides andsulfated fatty acid monoglycerides are also suitable.

The sulfonate-type surfactants are primarily the alkylbenzene sulfonatescontaining C₉₋₁₅ alkyl groups, sulfosuccinic acid monoesters anddiesters containing 6 to 22 carbon atoms in the alcohol components andthe esters of a-sulfofatty acids, for example the a-sulfonated methyl orethyl esters of hydrogenated coconut oil, palm kernel oil or tallowfatty acids. Other suitable surfactants of the sulfonate type are thealklane sulfonates obtainable from C₁₂₋₁₈ alkanes by sulfochlorinationor sulfoxidation and subsequent hydrolysis or neutralization or byaddition of bisulfite onto olefins and also olefin sulfonates, i.e.mixtures of alkene and hydroxyalkane sulfonates and also disulfonateswhich are obtained, for example, from long-chain monoolefins with aterminal or internal double bond by sulfonation with gaseous sulfurtrioxide and subsequent alkaline or acidic hydrolysis of the sulfonationproducts.

Bleaching agents are preferably selected from the type containingperoxygen, as hydrogen peroxide, alkali perborate, alkali percarbonate,alkali persilicate and/or alkali persulfate. Particularly preferred aresodium perborate monohydrate and sodium percarbonate. Bleaching agentsmay be present in amounts of 5% by weight to 25% by weight, moreparticularly 7% by weight to 20% by weight.

Bleach activator compounds include in particular N- or O-acyl compounds,for example polyacrylated alkylene diamines, more particularlytetraacetyl ethylene diamine, N-acrylated triazines, more particularly1,5-diacetyl-2,4-dioxohexahydro-1,3,5-triazine, acylated glycolurils,more particularly tetraacetyl glycoluril, N-acylated hydantoins,hydrazides, triazoles, urazoles, diketopiperazines, sulfuryl amides andcyanurates, also carboxylic anhydrides, more particularly phthalicanhydride, carboxylic acid esters, more particularly sodiumisononanoyloxy benzene sulfonate, and acylated sugar derivatives, moreparticularly pentaacetyl glucose. The bleach activator may be coated inthe usual way with shell-forming substances or may be granulated,optionally using granulation aids, and if desired may contain otheradditives, for example dye. A bleach activator which formsperoxycarboxylic acids with 2 to 12 carbon atoms, in particularperoxoacetic acid, under the washing conditions is preferably used. Aparticularly preferred bleach activator is tetraacetyl ethylene diamine(TAED) granulated with carboxymethyl cellulose with average particlesizes of 0.01 mm to 0.8 mm, which may be produced by the processdescribed in European Patent EP-B-0 037 026. In addition to the abovementioned bleach activators or even substituting them so-called bleachcatalysts may be used, which are transition metal complexes, for exampleas described in

Enzymes which may be present in the detergents according to theinvention, in addition to the cellulase according to the definition, areproteases, lipases, cutinases, amylases, pullulanases, other cellulases,hemicellulases, xylanases, oxidases and/or peroxidases. They may bepresent in amounts up to 5% by weight, preferably 0.2% by weight to 2%by weight.

The detergent compositions of the invention may be formulated in anyconvenient form e.g. as a powder or liquid. For the production ofdetergents with high apparent density of e.g. 650 g/l to 950 g/l amethod using an extrusion step, as described in European patent EP-B-0486 592, is preferred.

Fabric softening compositions comprising the inventive cellulase maycomprise further to this cellulase cationic surfactants, preferably ofthe so-called esterquat type, which are capable of fabric softening andwhich may increase the fabric softening properties of the compositions.

EXAMPLES Example 1 Production of Cellulases

Screening for cellulase producing microorganisms

Two methods were applied for the isolation of cellulase-producingmicroorganisms:

1) the soil and water samples were suspended in 0.85% saline solutionand directly used in the carboxymethyl cellulose (CMC)-agar diffusionassay for detection of cellulase producing colonies.

2) The soil and water samples were enriched for cellulase containingstrains by incubation in a cellulose containing liquid minimal medium orGAM-medium for 1 to 3 days at 40° C. Cultures that showed bacterialgrowth were analyzed for cellulase activity using the CMC-agar diffusionassay for detection of cellulase producing colonies.

Isolation of alkalitolerant, cellulase producing strains

Strains that showed clearing zones in the agar diffusion assay werefermented in 25 milliter GAM-medium in 100 milliliter shake flasks in anIncubator Shaker (New Brunswick Scientific, Edison, N.J., USA), at 250r.p.m. at 40° C. for 72 hours. CMCase activity was determined in theculture broth at pH 9 and 40° C.

Isolation of cellulase genes

Genomic gene libraries of the alkalitolerant cellulase producing strainswere constructed in plasmid pTZ18R (Mead, D. A., et al. (1986) ProteinEngineering 1, 67). Recombinant clones were screened by agar diffusionof CMC-agar as described by Wood, P. J., et al. (1988) Methods inEnzymology 160, 59-74. Strains that showed clearing zones around thecolony were isolated. The CMCase activity of the recombinant strains wasdetermined after fermentation for 48 hours at 30° C. in 4*YEP-medium.The plasmid DNA of the recombinant strains was isolated and the insertswere characterized by restriction enzyme analysis and nucleotidesequence analysis.

Media

The minimal medium (pH 9.7) using in the CMC-agar diffusion assay andthe enrichment procedure, consisted of KNO₃ 1%, Yeast extract (Difco)0.1%, KH₂PO₄ 0.1%, MgSO₄.7H₂O 0.02%, Na₂CO₃ 1%, NaCl 4% and 0.25% CMC(Sigma C-4888). For solidification 1.5% agar was added.

The complex medium (GAM) used for enzyme production of the donor strainsconsisted of Peptone (Difco) 0.5%, Yeast extract (Difco) 0.5%,Glucose.H₂O 1%, KH₂PO₄ 0.1%, MgSO₄.7H₂O 0.02%, Na₂CO₃ 1%, NaCl 4%. ThepH was adjusted to 9.5 with 4M HCl after which 1% CMC was added.

The complex medium (4*YEP) used for the enzyme production in E. colirecombinant strains consisted of Yeast extract (Difco) 4%, Peptone(Difco) 8%, lactose 0.2%, 100 μg/ml ampicilline.

CMC-agar diffusion assay for colonies

Cell suspensions in 0.85% saline solution were plated on CMC-containingminimal medium. After incubation for 1 to 3 days at 40° C., the plateswere replica plated and the parent plate was flooded with 0.1% Congo Redfor 15 minutes. The plates were destained with 1M NaCl for 30 minutes.The strains that showed a clearing zone aroung the colony were isolatedas potential cellulases producing microorganisms.

CMC-agar diffusion assay for liquid fractions

Aliquots of 40 μl of enzyme solution or fermentation broth were pipettedin wells punched out from a layer of 5 mm of minimal medium in a petridish. After incubation for 16 hours at 40° C. cellulase activity wasdetected by Congo Red/NaCl treatment. The diameter of the clearing zoneis a measure for the CMCase activity.

Resulting cellulase

These experiments resulted in the isolation of a cellulase producingmicroorganism which was deposited thereafter as CBS 670.93. Themicroorganism was classified as a new species of the genus Bacillus.Cloning experiments with the CBS 670.93 strain as a donor strainresulted in the isolation of an E. coli clone which was able to producea cellulase called BCE 103. The nucleotide sequence of the gene codingfor said cellulase was analysed. From the cellulase BCE 103 theN-terminal amino acid sequence was determined using standard methods forobtaining and sequencing peptides (Finlay & Geisow) (Eds.), ProteinSequencing—a practical approach, 1989, IRL Press). The amino acidsequence of the cellulase was deduced from the nucleotide sequence,using the N-terminal amino acid sequence for the starting point of themature protein.

The nucleotide sequence for BCE 103 is shown in SEQ ID NO. 1 and theamino acid sequence is shown in SEQ ID NO. 2.

Purification of the cellulase

After the fermentation the cells were separated from the culture liquidby centrifugation (8000 rpm). The cellulase in the supernatant wasprecipitated with ammonium sulphate (65% saturation). The precipitatewas dissolved in 25 mM phosphate buffer pH 7+5 mM EDTA until aconductivity of 7 mS/cm. This solution was applied to a Q-Sepharose FF(diameter 5 cm, length 10 cm) Anion Exchange column, after which thecolumn was washed with 25 mM phosphate buffer pH 7+5 mM EDTA until anabsorbency of 0.2 AU. A gradient of 0 to 0.5 M NaCl in 25 mM phosphatepH 7 was applied to the column in 80 minutes followed by a gradient from0.5 to 1 M NaCl in 10 minutes. Depending on which cellulase was appliedto the column, elution took place in the first or the second gradient.After elution the column was cleaned (upflow) with 1 M NaOH andequilibrated again with 25 mM phosphate pH 7+5 mM EDTA. Depending on theelution the obtained cellulase had a purity of up to about 80%.

Characterization

CMC'ase assay

Assays for cellulase activity were performed using modified methods ofthe PAHBAH method (Lever M. Anal. Biochem. 1972, 47, 273-279 and LeverM. Anal. Biochem. 1977, 81, 21-27).

Procedure

A test tube is filled with 250 μl 2.5% CMC in 50 mM glycine buffer pH 9(CMC-low viscosity is purchased from Sigma) and 250 μl aliquotscellulase, diluted in the appropriate buffer. The test tube is incubatedfor 30 minutes at 40° C. in a waterbath, whereafter 1.5 ml of a dailyfresh prepared PAHBAH solution (1% PAHBAH in 100 ml 0.5 M NaOH with 100μl bismuth solution (containing 48.5 g bismuth nitrate, 28.2 g potassiumsodium tartrate and 12.0 g NaOH in 100 ml) is added. The mixture isheated at 70° C. for 10 minutes, after which it is cooled on ice for 2minutes. The absorption is measured at 410 nm. To eliminate thebackground absorbance of the enzyme samples a control experiment isexecuted as follows: a tube with substrate is incubated under the sameconditions as the test tube. After the incubation 1.5 ml PAHBAH and theenzyme preparation is added (in this order). One unit (U) is defined asthe amount of enzyme producing 1 μmol of glucose from CMC equivalentdetermined as reducing sugars per minute per gram product.

The buffer used for the determination of the pH/temperature profiles isa phosphate/citrate system. The pH/temperature profiles were determinedusing a fixed enzyme concentration which fits in the linear range of thedose response profile measured at pH 7 and 40° C. This enzymeconcentration was used for the measurement of the activities under allother determined conditions.

The results for the cellulase BCE 103 are shown in FIG. 1. Thiscellulase shows good activities at alkaline pH, which makes it suitablefor application in detergents with an alkaline pH.

Example 2

Similar procedures starting with the alkalophilic bacillus strain CBS669.93 resulted in cellulase BCE 113. The results for this cellulase BCE113 are shown in FIG. 2. This cellulase also shows good activities atalkaline pH, which makes it suitable for application in detergents withan alkaline pH.

Example 3 Measurement of Tensile Strength and Antipilling

As described for the evaluation of TSL, washing and experiments wereperformed using as detergent matrix a Colour Detergent without bleach,without perfume and enzymes (105 g detergent per wash cycle, pH10.5), aswashing machine a type Miele® W 717, temperature 40° C., program_(“)Normalprogramm”, with water of a hardness of 16° dH (Germanhardness), wash load 3.5 kg. 25 washes.

Experiments using a composition according to the invention (D1) as wellas comparisons (C1 to C3) were run in parallel in identical machines:

C1: detergent matrix without cellulase

C2: detergent matrix+0.288 mg endoglucanase V from Humicola insolens

C3: detergent matrix+cellulase mixture from Humicola insolens sold asgranules Celluzyme® 0.7T

D1: detergent matrix+0.288 mg cellulase BCE 103

D2: detergent matrix+0.288 mg cellulase BCE 113

TABLE 1 Results of TSL-measurements [%] Composition TSL C1 0 C2 100 C338 D1 12

Using washing machines of type Miele® W 914, under otherwise identicalconditions, gave the following results:

TABLE 2 Results of TSL-measurements [%] Composition TSL C1 0 C2 100 D20.6

Example 3 Measurement of Antipilling and Calculation of the Ratio TSL toAP

The evaluation of antipilling properties was done with increasedconcentrations of cellulases for better quantitative evaluation of theeffect. A Colour Detergent (5 g/l, 10 wash cycles at 40° C.) with theaddition of cellulase as given in Table 3 was used on _(“)pilled” sweatshirt cotton material (washed 25 times at 60° C. with a detergentwithout cellulase). Evaluation of the pilling was done with the opticalmeasurement system as described before; a degree of pilling of 0% wasassigned to the _(“)pilled” material.

TABLE 3 Results of AP-measurements [%] degree of pilling Enzymeconcentration EG V BCE 103 AP [%]of BCE 103 25 μg/ml −12.8% −8.4% 65%37.5 μg/ml −16.0% −9.6% 59% 50 μg/ml −22.8% −15.6% 68%

An average AP of 64% can be calculated for BCE 103 cellulase. BCE 113cellulase showed under the same conditions an average AP of 100%.

Using the values for TSL in Tables 1 and 2, the ratios of TSL to AP forthe various cellulases are as in the following Table 4:

TABLE 4 Ratio TSL to AP Enzyme Ratio EG V   1 BCE 103 ≈0.2 BCE 113 ≈0.02

Example 4 Further Test Procedures

Anti redeposition test

20 ml 50% pigmented soil (fresh prepared, daily and consisting of 86%kaolin, 8% soot (Flammruss 101, obtained from Degussa AG), 4% iron oxideblack and 2% iron oxide yellow (from Henkel Genthin GmbH)), in adetergent (Persil color® without enzymes, 5 g/l, pH 8.5) was, underagitating (90 ppm) incubated with white cotton fabric (prewashed, 5 cmdiameter, obtained from Windelbleiche, Krefeld). Cellulase was addeduntil a final concentration of 1 mU/ml. The mixture was incubated for 30minutes at 40° C., 90 rpm. As a control the same incubation was carriedout without the addition of cellulase. After the incubation the fabricwas rinsed thoroughly with running cold water. After drying thewhiteness of the fabric was measured by remission (4 measurements perfabric) using a Micro colour Dr. Lange® Colourimeter. The control valuewas substracted from the sample value. The results, expressed as deltaRem, are shown in Table 5.

Fibre Damage Test

One pad of cotton wool (100% cotton, Warenhandels GmbH, Buchholz, MarkeOlivia, Selling agency: Aldi) was incubated in 40 ml wash liquor (Persilcolor® without enzyme, 5 g/l pH 8.5), cellulase at a final concentrationof 1 mU/ml was added in a sealed flask and incubated for 20 hours at 40°C. under agitation (90 rpm). After the incubation, fibre damage wasmonitored by the measurement of the quantity of the reducing sugars insolution, using the PAHBAH method described in Example 1. As a controlthe same incubation was carried out without the addition of cellulase.The results are shown in Table 5.

Adsorption Test

White cotton fabric (Windelbleiche, Bielefeld) prewashed with germanPersil® without enzymes at 60° C., was cut round to 9 cm diameter(approx. 0.920 gram). One cotton swatch was incubated in 50 ml 50 mMglycine-NaOH buffer pH 9 including 0.1% SDS and 1 ml cellulase sample(600 mU/ml) for 60 minutes at 30° C. 2 ml samples were taken at T=0 andat T=60 minutes and were diluted directly (1:2) with 50 mM MES-buffer pH6.5 and stored at 4° C. until measurement. As control the sameincubation was carried out without the addition of cotton textile. Theactivity measurement was determined with a PAHBAH method as described inExample 3, but at pH 6.5 in 50 mM MES buffer. The adsorption wasexpressed as relative adsorption where the activity applied at the startof the experiment was set as 100%, T=0. 100% activity value−remainingactivity (%)=adsorption (%). The results are shown in Table 5.

TABLE 5 Results of the Antiredeposition Test, Fibre Damage Test andAdsorption Test Antiredeposition Fibre Damage Enzyme [detta REM] [mU]Adsorption [%] BCE 103 5.0 0.025 7 KAC ®^(a)) 7.5 0.006 0 EG V 1.2 0.15536 ^(a))Cellualse of Kao Corporation

Cellulase BCE 113 performed in theses tests at least as well ascellulase BCE 103.

Softening test

The softness of fabrics treated as in Example 3, but after 15 washcycles, was rated by an expert panel (5 persons) who awarded gradesbetween 0 (fabric washed 25 times with a detergent without cellulase)and 6 (fabric prior to any wash) by the feel of the fabrics.Compositions as defined in Example 2 were used in the washings. Theaverage rates are given in Table 6. It can be seen that the compositionsaccording to the invention showed the best performance.

TABLE 6 Results of the Softening Test Composition Rate C1 0 C2 2.1 C31.5 D1 2.3 D2 2.2

TABLE 6 Results of the Softening Test Composition Rate C1 0 C2 2.1 C31.5 D1 2.3 D2 2.2

What is claimed is:
 1. A laundry detergent composition consistingessentially of 0.8 ppm to 80 ppm of a cellulase, wherein the cellulasehas a ratio of tensile strength loss to antipilling properties of lessthan
 1. 2. A laundry detergent composition according to claim 1consisting essentially of 1 ppm to 40 ppm of the cellulase.
 3. A methodof laundering cotton-containing fabrics comprising contacting acotton-containing fabric with a cellulase, wherein the cellulase has aratio of tensile strength loss to antipilling properties of less than 1,wherein the fabric is contacted with an aqueous laundering solutionconsisting essentially of 0.01 mg/l to 0.2 mg/l of the cellulase.
 4. Amethod according to claim 3, wherein the aqueous laundering solutionconsists essentially of 0.015 mg/l to 0.1 mg/l of the cellulase.